Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis

Abstract

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.

Exploiting the Achilles’ heel of membrane trafficking in trypanosomes

“…Further analyses revealed that aquaglyceroporin 2 ( AQP2 ) determines MPXR [44•, 45•]. Further, re-introduction of a wild-type TbAQP2 allele in even the most resistant strains fully restores drug sensitivity [45•, 46••, 47•, 41]. Indeed, expressing TbAQP2 in Leishmania mexicana promastigotes profoundly increases their sensitivity to both pentamidine and melarsoprol [45 • ].…”

“…There is much scope for exploiting these drug uptake mechanisms to deliver new drugs, especially given the surprising variety of compounds that they internalize. Notably, drug-loaded nanoparticles, coated with a nanobody specifically targeting the trypanosome surface, can be used to bypass the usual delivery route altogether and deliver drugs via endocytosis [47 • ]. Indeed, this approach actually increases the efficacy of pentamidine, and a similar approach is likely to be effective for delivering other drugs.…”

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enterTrypanosoma brucei

“…There is overwhelming consensus that expression of TbAQP2 is associated with the extraordinary sensitivity of T. brucei to pentamidine and melaminophenyl arsenicals, and that mutations and deletions in this locus cause resistance (Baker et al, 2012, 2013; Graf et al, 2013, 2015, 2016; Pyana Pati et al, 2014; Munday et al, 2014, 2015a; Unciti-Broceta et al, 2015). What has remained however unclear is the mechanism underpinning these phenomena – there are currently no documented other examples of aquaporins transporting such large molecules..…”

“…The endocytosis model identifies only two key residues for pentamidine access (Leu264) and binding (Asp265) in TbAQP2 (Song et al, 2016). Yet, multiple clinical isolates and laboratory strains contain chimeric AQP2/3 genes associated with resistance and/or non-cure that have retained those residues and should thus allow binding and internalisation of pentamidine (Graf et al, 2013; Pyana Pati, 2014; Unciti Broceta et al, 2015; Munday et al, 2014). Although we find that introduction of the AQP3 Arg residue in position 264 (TbAQP2 L264R ) disables pentamidine transport, this is because the positively charged arginine, in the middle of the pore, is blocking the traversing of all cations through the pore, as is its common function in aquaporins (Beitz et al, 2006; Wu et al, 2009).…”

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance inTrypanosoma brucei

“…Melarsoprol-pentamidine cross-resistant strains and field isolates from relapse patients all possess mutations at the locus encoding TbAQP2, including deletions, single nucleotide polymorphisms and chimerisation [27–30]. Pentamidine-resistant trypanosomes from a cohort of relapse patients from the Democratic Republic of Congo also have TbAQP2 chimerisation with the coding sequence for the C-terminal trans- membrane domain replaced by TbAQP3 and in most cases without altering the selectivity filter characteristic of TbAQP2 (NSA/NPS – IVLL motifs) [29,31,32].…”